Pertussis toxin (PTX) is an oligomeric protein produced by Bordetella pertussis, the etiologic agent of whooping cough or pertussis. PTX exerts a diverse array of biological effects both in vivo and in vitro and is thought to occupy a central role in the pathogenesis of pertussis. Importantly, PTX has been demonstrated to be an important component of acellular vaccines. PTX appears to exert many, if not most, of its pathobiological effects through the NAD-dependent ADP-ribosylation of guanine nucleotide-binding regulatory proteins of the membrane-bound adenylate cyclase complex of mammalian cells; therefore, PTX belongs to the group of ADP-ribosylating bacterial protein toxins , that include cholera toxin, the Escherichia coli heat-labile toxins, diphtheria toxin and Pseudomonas exotoxin A. Development of new and safer whooping cough vaccines is a major priority of health vaccine manufacturers. Evidence accumulating from clinical trials of acellular pertussis products strongly indicates that pertussis toxin will be a necessary, and perhaps sufficient component of any new vaccine. Anticipating the need for purer and non-reactogenic and recombinantly expressed the genes for each of the toxin component proteins in Escherichia coli. This project involves purification and characterization of these protein antigens produced by the host system. In many instances, these expressed proteins represent up to 40% of the total cell protein; however, these proteins are often insoluble and form inclusion bodies in the E. coli cell. Various techniques were developed to solubilize these proteins. We have succeeded in purifying various versions of subunits SI, S2, and S4. Manipulation of the gene segment encoding the A protomer (SI subunit) has permitted identification of regions possessing: both immunodominant and immunoprotective epitopes as well as sites critical for enzymatic activities. We expect that our recombinant reagents will allow us to develop a stable, safe, and efficacious acellular vaccine against pertussis.